ARF Locus

The INK4a/ARF Locus DE Quelle, University of Iowa, Iowa City, IA, USA and University of Iowa Hospitals and Clinics, Iowa City, IA, USA J Nteeba, University of Iowa, Iowa City, IA, USA BW Darbro, University of Iowa, Iowa City, IA, USA and University of Iowa Hospitals and Clinics, Iowa City, IA, USA r 2016 Elsevier Inc. All rights reserved.

Introduction The cell cycle is a series of ordered biochemical events resulting in division of a mother cell into two identical daughter cells. This process requires the accurate duplication of DNA and equal segregation of chromosomes into each daughter cell, and it must be tightly regulated throughout the life span of complex organisms in order to prevent the unrestrained proliferation of damaged cells (i.e., cancer). Proper cell cycle progression is monitored by checkpoints, surveillance mechanisms that ensure the completion of each cell cycle phase and maintenance of genome integrity (Malumbres and Barbacid, 2009; Sherr, 2004). They are enforced by tumor suppressor proteins that arrest the cell cycle, enabling time for the cell to repair mistakes before reentering the cycle, or eliminate cells (e.g., by programmed cell death (apoptosis)) if the damage is intolerable. Two of the most important tumor suppressors are the retinoblastoma (RB1) and p53 proteins, which are activated by the products of the INK4a/ARF locus, p16INK4a and the alternative reading frame protein (ARF), respectively (Levine and Oren, 2009; Sherr, 2001). This review takes a historical look at the discovery and study of INK4a/ARF. It begins with a consideration of fundamental mechanisms controlling the cell cycle, highlighting where and how p16INK4a and ARF exert their anti-proliferative effects, followed by an analysis of their individual and interconnected roles in cancer. Growing evidence that the INK4a/ARF locus plays a pivotal role in other important aspects of normal biology will also be discussed.

numerous genes required for DNA replication, permitting cells to enter and progress through S phase. Interestingly, RB1 has two relatives, p107 and p130, that act similarly although their tissue distribution varies and only RB1 is commonly mutated in human cancers (Burkhart and Sage, 2008). G1 phase cyclins (principally D and E) and their partners (Cdks 2, 4, and 6) are rate-limiting in promoting cell cycle progression. Their overexpression in cultured cells shortens G1 phase and accelerates S phase entry, whereas their loss, depending on the cell type, prevents cell cycle progression (Sherr and Roberts, 2004). Moreover, many human cancers have gene amplifications, mutations, or translocations that elevate the expression and activity of these cyclins and Cdks (Malumbres and Barbacid, 2009). Mimicking those effects in mice causes tumor development in vivo. For example, artificial overexpression of cyclin D1 in mammary epithelial cells using the mouse mammary tumor virus promoter (MMTV) causes breast cancer in MMTV-D1 transgenic mice (Wang et al., 1994). Conversely, mice lacking cyclin D1 are protected from breast cancer in animals with activated Ras or Her2/neu/ErbB2 oncogenes (Lee and Sicinski, 2006). Such studies have established that elevated cyclin D-Cdk4/6 and E-Cdk2 activities are key drivers of tumorigenesis. As a result, investigators have sought to understand the mechanisms that normally restrict cyclin D-Cdk4/6 and ECdk2 kinase activities in non-transformed cells and define how Mitogens

Control and Significance of G1–S Phase Progression in Mammals The 1990s was a time of rapid advancement in our understanding of the mammalian cell cycle, particularly the events controlling the first Gap period (G1 phase) and mechanisms by which cells begin DNA synthesis (S phase). Building of earlier studies of cyclins and cyclin-dependent kinases (Cdks) in yeast and Xenopus, we learned that the mammalian D-type cyclins (D1, D2, and D3) are regulatory subunits whose expression is maximally induced by mitogens in mid-G1 phase, at which time they form active serine/threonine kinases with their catalytic partners, Cdk4 or Cdk6 (Hunter and Pines, 1994; Malumbres and Barbacid, 2009; Sherr and Roberts, 2004). Those holoenzymes promote G1 to S phase progression by phosphorylating and inactivating the RB1 tumor suppressor protein (Figure 1(a)). RB1 phosphorylation, first by cyclin D-Cdk4/6 followed by cyclin E-Cdk2 and cyclin ACdk2 kinases, results in the release of E2F transcription factors from inhibitory RB1 complexes (Harbour and Dean, 2000). Once free, activated E2F proteins stimulate the expression of

Encyclopedia of Cell Biology, Volume 3

doi:10.1016/B978-0-12-394447-4.30060-8

Oncogene activation, aging signals

K4

D1

INK4a D1

P

E2F

RB1

Cell proliferation

E2F Inactive

Inactive

(a)

K4

S phase genes

P

+

RB1

+

(b)

G1 arrest or senescence

Figure 1 The INK4a–RB1 anti-proliferative pathway. (a) The upregulation of cyclin D1 by mitogens promotes formation of D1– Cdk4 kinases. Phosphorylation and inactivation of RB1 enables E2F to bind DNA and transcribe genes required for S phase entry and cell proliferation. (b) p16INK4a expression is induced by hyperproliferative signals from activated oncogenes or events during cellular aging that lead to INK4a derepression. Cdk4 binding by p16INK4a disrupts D1–Cdk4 association and leads to accumulation of hypophosphorylated RB1 and inactive E2F. Cells become reversibly arrested in G1 phase or undergo a permanent arrest termed senescence.

447

448

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus

those events are compromised in human tumors. Summarizing over 20 years of research, studies show that the changing phosphorylation status of each cyclin and Cdk subunit plays an essential role in controlling their subcellular localization, stability, conformation, and binding to each other. Disrupting any of those controls can promote tumor formation by enhancing the activity of cyclin D- and E-dependent kinases. An additional level of negative control involves small proteins that bind and inhibit those kinases. These cyclin-dependent kinase inhibitors (CKIs) fall into two major classes, the Cip/ Kip and INK4 proteins, and their in-depth characterization has been motivated by evidence that their inactivation occurs in a significant fraction of human cancers and promotes tumorigenesis (Malumbres and Barbacid, 2009). The Cip/Kip family (‘Cdk inhibitory’ or ‘Kinase inhibitory’ proteins) includes p21Cip1 (CDKN1A), p27Kip1 (CDKN1B), and p57Kip2 (CDKN1C). Low levels of these proteins are actually critical for cyclin D-Cdk4/6 assembly and cell cycling, and their sequestration by D-Cdk4/6 complexes permits activation of the Cdk2 holoenzymes (Sherr and Roberts, 2004). However, their elevated expression strongly inhibits cyclin D-dependent kinases and halts cell proliferation, whereas even low concentrations of Cip/Kip proteins inhibit cyclin E-Cdk2 and A-Cdk2 as well as the M phase kinase, cyclin B-Cdk1. While Cip/Kip factors act broadly, the INK4 proteins selectively bind Cdk4 (or its homolog, Cdk6) and act as specific ‘Inhibitors of Cdk4’ (Figure 1(b)). The four members of the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) were named based on their size (kDa) and order of discovery. These proteins are defined by the presence of four ankyrin repeats, which are required for their Cdk binding and inhibitory activity.

p16INK4a and RB1-Mediated Senescence p16INK4a is the first identified, prototypical member of the INK4 family (Serrano et al., 1993). p16INK4a and the other INK4 proteins each bind and inhibit Cdk4 (or Cdk6), and when expressed in vivo they primarily exist in binary INK4– Cdk complexes. Structural studies of p16INK4a and p18INK4c showed they function by blocking ATP binding and distorting the cyclin binding site in the Cdk, effectively preventing or disrupting the formation of cyclin D-Cdk4/6 holoenzymes (Jeffrey et al., 2000; Russo et al., 1998). In proliferating cells, the INK4 proteins are either absent (p16INK4a) or expressed at very low levels (e.g., p18INK4c). However, their expression is greatly increased in response to various cellular stresses or antiproliferative signals, such as TGF-β, and their consequent inhibition of D-Cdk4/6 kinases potently arrests the cells in G1 phase. Importantly, the INK4 proteins have no growth inhibitory activity in cells that lack functional RB1, illustrating the strict linearity of the INK4–cyclin D-Cdk4/6–RB1 pathway (Sharpless and DePinho, 1999). The primary differences between p16INK4a and its relatives occur in their tissue distribution, signals regulating their expression, and role in biology. For instance, p18INK4c and p19INK4d are normally induced by differentiation and expressed in non-proliferating cells within various tissues (e.g., spermatocytes in the testes and sensory hair cells in the

corti) (Zindy et al., 1997; Krishnamurthty et al., 2004). Their expression is vital for maintaining a quiescent state of differentiated cells, which has dire consequences when lost as indicated by the sterility of p18INK4c/p19INK4d-null male mice and deafness of p19INK4d-null animals (Zindy et al., 2001; Chen et al., 2003). By comparison, p16INK4a is undetectable in embryos and young mammals and becomes increasingly expressed in aging mice and humans (Zindy et al., 1997; Krishnamurthty et al., 2004; Ressler et al., 2006). That expression pattern correlates with p16INK4a playing a role in cellular senescence, an irreversible cell cycle withdrawal associated with cell and organismal aging. Senescence is also important for tumor suppression, not only by promoting the permanent arrest of damaged cells but also by inducing their immune clearance (Xue et al., 2007). p16INK4a is upregulated in cells undergoing senescence, induces telomere-independent senescence when expressed, and is required for senescence in cultured human cells (Darbro et al., 2005; Collado et al., 2007). Since loss of senescence is a key step in tumorigenesis, it is not surprising that INK4a gene inactivation is seen in nearly half of all human tumors (Ruas and Peters, 1998). While most of the INK4 and Cip/Kip proteins (except p19INK4d) have tumor suppressive activity, the frequency of INK4a inactivation in cancers far exceeds that of the other CKIs (Malumbres and Barbacid, 2009), suggesting a more prominent contribution of INK4a to tumor suppression.

INK4a/ARF – a Dual Coding Locus in Mammals The role of INK4a in cancer became clouded by the startling realization that another transcript is encoded by the locus. Several groups made this discovery at the same time (Stone et al., 1995; Mao et al., 1995; Duro et al., 1995; Quelle et al., 1995), and while some thought the alternate transcript was noncoding it became clear that a protein was generated and it was a powerful cell cycle inhibitor in its own right (Quelle et al., 1995). The locus is unusual because it encodes two unrelated proteins derived from separate first exons, exon 1α for p16INK4a and exon 1β for ARF, that are spliced to the same second exon (Figure 2). Each first exon supplies its own ATG start codon that initiates translation in alternative reading frames through shared sequences in exons 2 and 3, hence the name ‘ARF’ for the product of the β transcript. The resulting p16INK4a and ARF (mouse p19ARF, human p14ARF) proteins share no amino acid identity, and their structures and mechanisms of action are entirely distinct. While p16INK4a acts in the RB1 pathway, ARF activates the p53 tumor suppressor as well as other cancer pathways (Sherr, 2006; Kim and Sharpless, 2006; Gil and Peters, 2006), as described in following sections. This dual coding region was then renamed INK4a/ARF although the original locus designation, CDKN2A (which fails to convey the locus complexity), is still used prevalently in databases. The INK4a/ARF locus is interesting from an evolutionary perspective. It is conserved in mammals, but chickens lack INK4a and only express a truncated ARF protein (p7) from an orthologous exon 1β while neither INK4a nor ARF are found in fish (Gil and Peters, 2006). However, fish and chickens do retain an INK4b gene, which in the mammalian genome

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus

p15INK4b

1

2

ANRIL (CDKN2BAS)

449

p16INK4a

1

1

2

3

p19ARF (mouse) p14ARF (human)

Figure 2 Schematic of the INK4a/ARF locus. The ARF and INK4a genes, depicted with green and blue exons, respectively, span B20 kb on human chromosome 9p21.3 or mouse chromosome 4. The α transcript for p16INK4a (blue) and β transcript for ARF (green) are generated from distinct promoters upstream of their first exons. Mouse and human ARF proteins differ in size due to C-terminal variations and are either 19 kDa (mouse) or 14 kDa (human). The INK4b gene (gray exons) encoding p15INK4b resides centromeric to exon 1β of ARF. Intronic sequences are represented by the dashed black horizontal line. Representative exons (thought to total 19 and spanning B120 kb) of the long noncoding RNA, ANRIL (also called CDKN2BAS), are depicted with red ovals and the transcript shown as a red arrow. ANRIL is transcribed in an antisense direction from a promoter element near that for exon 1β of ARF. This image is not drawn to scale, but to give some perspective exon 1β resides B8 kb from p15INK4b exon 2 and B13 kb from exon 1α. Notably, this locus is a genomic hotspot for single nucleotide polymorphisms (SNPs) associated with several cancers and a variety of other age-related diseases in humans.

resides proximal to ARF exon 1β (see Figure 2). Based on sequence analyses across species, it is thought that INK4a arose fairly recently in evolution by duplication of INK4b followed by subsequent acquisition of exon 1β. The more recent appearance of ARF is consistent with the notion that dual coding allows for the expression of novel, intrinsically disordered proteins that perform new functions (Kovacs et al., 2010). Indeed, p16INK4a has a well-defined α-helical structure and binds Cdk4/6 (Byeon et al., 1998), while ARF is largely unstructured (especially in its C-terminal half derived from the alternative reading frame of exon 2) and participates in other pathways (Kovacs et al., 2010; Sherr, 2006). Dual coding of unrelated proteins from overlapping reading frames is typical in the compact genomes of prokaryotes and viruses, but it is rare in eukaryotes. For years, INK4a/ARF was thought to be the only genuine dual coding gene in mammals but other loci of biological importance have been identified. For instance, the XBP1S transcription factor and its inhibitor, XBP1U, arise from alternative reading frames in the XBP1 (X-box binding protein 1) gene and their coordinated activity is required for such fundamental processes as plasma cell differentiation and the unfolded protein response (Yoshida et al., 2001; Iwakoshi et al., 2003). Likewise, GNAS1 (G protein alpha subunit) encodes the unrelated G proteins, XLαs and ALEX, whose interaction is critical for multiple signaling pathways and whose loss of function is linked with several human disorders (Freson et al., 2003; Turan and Bastepe, 2013; Klemke et al., 2001). An intriguing theme is that the dual products perform related tasks, either by binding and regulating each other (e.g., XBP1 and GNAS1) or by performing common functions (e.g., tumor suppression by INK4a/ARF). At least 40 additional dual coding genes, which have been dubbed hallmarks of fascinating biology, are thought to exist in eukaryotes (Kovacs et al., 2010; Chung et al., 2007).

INK4a/ARF – One Locus, Two Important Tumor Suppressors Chromosome 9p21 where INK4a/ARF resides is the second most frequently inactivated locus in human cancers (Ruas and Peters, 1998). Since their discovery, the relative role of

p16INK4a versus ARF in cancer has been a matter of intense investigation and debate. The cumulative data show that INK4a and ARF are most often co-inactivated by silencing or homozygous deletion of the entire locus, frequently including INK4b. In some cancers, such as glioblastoma, loss of INK4a/ ARF is almost always achieved by gene deletion whereas in other tumor types, such as renal cell carcinomas, promoter methylation and silencing is the predominant mechanism (Figure 3). Inactivating mutations (nonsense, missense, and frameshift) of the locus are also common in certain cancers, especially sporadic and familial melanoma, as well as stomach and pancreatic adenocarcinomas. Notably, The Cancer Genome Atlas (TCGA) and other databases compile tumor data (copy number, mutation, methylation, RNA sequencing, etc.) for CDKN2A, which fails to distinguish between INK4a- and ARF-specific alterations unless data for each gene in each tumor are carefully examined. Such analyses reveal that ARF is selectively disabled in various cancers by alterations of exon 1β or ARF’s open reading frame in exon 2 (Ozenne et al., 2010; Maggi et al., 2014). Nonetheless, the vast majority of tumor mutations specifically target p16INK4a sequences in exons 1α and 2 (Kim and Sharpless, 2006; Gil and Peters, 2006). This fostered a lingering perception that p16INK4a would play a larger role in cancer than ARF. Specific disruption of exon 1β or exon 1α in mice conclusively showed that both ARF and p16INK4a are tumor suppressors (Kamijo et al., 1997; Kamijo et al., 1999; Sharpless et al., 2001, 2004; Krimpenfort et al., 2001). In fact, ARF-null mice develop spontaneous tumors with a slightly shorter latency than INK4a-null animals (Table 1). ARF-null mouse embryo fibroblasts (MEFs) also evade replicative and Ras oncogene-induced senescence, unlike MEFs lacking INK4a, indicating that ARF loss removes key checkpoints that normally prevent cell immortalization and transformation. The fact that p16INK4a loss had no effect on MEF senescence was perplexing given its apparent importance in human cell senescence (Kim and Sharpless, 2006; Campisi, 2005). An explanation was provided by studies of INK4a/ARF and INK4a knockout mice that lacked or retained INK4b, which showed p15INK4b is upregulated in the absence of p16INK4a and can compensate for its loss (Krimpenfort et al., 2007). Redundancy among the INK4 proteins is also illustrated by the robust

450

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus

90 Silenced Mutation

80

Deletion Amplification

70 Alteration frequency (percent of tumors)

Multiple alterations 60

50

40

30

20

10

+ + + + + + + + + + + + + + – + + + + + + + + + + + + + + + + + + – + + – – + + + – + + + + + + + + + + + + – – + – – + + – –

He

GB M ad Blad (TC GA an der d ) ( Me neck TCG lan (TC A) om a ( GA) TC P GA Lu ng anc N C ) sq rea ua s ( I-60 mo TC us G (TC A) GA Sto ) m C Lu ng ach CLE (TC ad GA Bla eno ) ( dd er TCG DL (MS A) Me BC KC l L Lu anom (TC C) ng G a (B A) ad Sa eno road) E rcom (Bro He soph a (T ad) a ad C an gus ( GA) dn Bro ec ad k ) AC (Bro C ( ad) TC pR C G Pro C (T A) CG sta Pa t nc e (M A) rea ICH s( ICG ) C)

0 Mutation data CNA data mRNA data

Figure 3 Frequency of genetic alterations at the CDKN2A locus in selected human malignancies. The relative frequency of gene deletion, mutation, silencing, and amplification occurring at the CDKN2A locus harboring INK4a and ARF is displayed for various cancers. The percentage of tumors with silencing of the locus was calculated based on the difference between the percent of tumors showing reduced CDKN2A mRNA (primarily obtained from RNA-Seq data but also some microarray results) and those with gene deletion. A z-score threshold of þ /  0.5 was used to determine if CDKN2A mRNA expression was down-regulated. Separate data sources interrogating the same tumor type can display different types and frequencies of CDKN2A genetic inactivation (e.g., compare results from two separate sources for melanoma, pancreatic, or bladder cancers). Such variation may reflect differences in patient populations examined (number, ethnicity, etc.) or simply that some studies solely examine copy number aberrations (CNA data) or sequence level mutation data (Mutation data), as indicated, with or without accompanying mRNA data. This graphic is derived from multiple data sources (listed) including TCGA (The Cancer Genome Atlas) and was compiled with the aid of the cBioPortal (Gao et al., 2013; Cerami et al., 2012). NCI-60, cell line database compiled by the U.S. National Cancer Institute; CCLE, Cancer Cell Line Encyclopedia; MSKCC, Memorial Sloan Kettering Cancer Center; Broad, Broad Institute of MIT and Harvard; MICH, Michigan Center for Translational Pathology; ICGC, International Cancer Genome Consortium; GBM, glioblastoma multiforme; Adeno, adenocarcinoma; ACC, adenoid cystic carcinoma; and pRCC, papillary renal cell carcinoma.

tumor phenotype (and lack of MEF senescence) in mice expressing a Cdk4 mutant (R24C) that fails to bind all INK4 proteins and escapes their inhibition (Sotillo et al., 2001a, b; Rane et al., 2002). Thus, ARF and p16INK4a are important

regulators of cellular proliferation and senescence whose loss facilitates cancer. Interestingly, tumor predisposition is enhanced in mice lacking both p16INK4a and ARF relative to the single

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus Table 1

451

Phenotypes of ARF, INK4a, and INK4a/ARF deficient or transgenic micea

Genotype

Alteration

Tumor phenotype

References

INK4a/ARF-/-

Exon 2 and 3 deletion

Serrano et al. (1996)

ARF-/-

Exon 1β deletion

INK4a-/-

Exon 1α deletion

INK4amt/mt

Knock-in of exon 2 point mutantb (encodes a defective p16INK4a protein)

Cdk4R24C/R24C

Knock-in of Cdk4 mutant insensitive to INK4 proteinsb Deletion of exon 1α, 1β, and INK4b exons plus INK4a exon 2 point mutantb Exon 1β deletion; Myc transgene in B cells

Sarcomas and lymphomas within 8.5 months; MEFs divide rapidly, are immortal and transformed by oncogenic Ras Sarcomas, lymphomas, and carcinomas within 9.5 months; MEFs divide rapidly, are immortal and transformed by oncogenic Ras Sarcomas and lymphomas within 11 months; increased T cell proliferation; MEFs senesce, not transformed by oncogenic Ras Low incidence of spontaneous tumors (B-cell lymphoma) within 17 months; melanomas develop upon loss of one allele of ARF; MEFs senesce and not transformed by oncogenic Ras Various tumors with complete penetrance, including invasive melanoma; MEFs immortal and easily transformed More tumor prone and wider tumor spectrum than INK4a/ARF-/- mice; MEFs more sensitive to oncogenic transformation than INK4a/ARF-/- cells Accelerated B-cell lymphomagenesis relative to Em-Myc micec; mice die by 7 weeks of age versus 6 months in Em-Myc mice Cutaneous melanoma with short latency and high penetrance Much broader spectrum of tumors, and multiple tumors per animal, compared to mice lacking ARF or p53 alone Accelerated formation of pancreatic neuroendocrine tumors with increased angiogenesis relative to RIP-Tag mice Accelerated development of pancreatic intraepithelial neoplasias (PanINs); metastatic pancreatic ductal adenocarcinoma (PDAC) and death by 2–3 months of age Development of PDGFB-induced gliomas; much greater incidence of high-grade tumors in ARF-/- mice than INK4a-/- mice

Weber et al. (2000)

Increased cancer resistance, reduced production of sperm, normal aging Further enhanced cancer resistance, absence of sperm, decrease in aging markers, and extended longevity

Matheu et al. (2004)

INK4b-/-; INK4amt/mt; ARF-/-

ARF-/-; Em-Myc

INK4a/ARF-/-; Tyr-RasG12V

ARF-/-; p53-/-; Mdm2-/-

ARF-/-; RIP-Tag

Pdx1-Cre; KRasG12D; INK4a/ ARFflox/flox

ARF-/- or INK4a-/-on Ntv-a or Gtv-a background

INK4/ARF-tg INK4/ARF-tg/tg

Exon 2/3 deletion; mutant KRas in melanocytes Triple knockout of ARF exon 1β, p53 and Mdm2 Exon 1β deletion; SV40 T antigen expression in β cells Tissue-specific expression of mutant KRas and INK4a/ARF exon 2/3 deletion Neural- or astrocytespecific expression of PDGFB in exon 1β or 1α deleted mice One extra allele of INK4b-ARF-INK4a Two extra alleles of INK4b-ARF-INK4a

Kamijo et al. (1997, 1999)

Sharpless et al. (2001)

Krimpenfort et al. (2001)

Sotillo et al. (2001a, b), Rane et al. (2002) Krimpenfort et al. (2007)

Eischen et al. (1999)

Chin et al. (1997)

Ulanet and Hanahan (2010)

Aguirre et al. (2003)

Tchougounova et al. (2007)

Matheu et al. (2009)

a

Incomplete listing of mouse models with altered INK4a and ARF. Naturally occurring point mutations found in human tumors, including melanomas. c Loss of INK4a or RB1 does not accelerate Em-Myc lymphomagenesis (Lowe and Sherr, 2003). b

knockout animals (Sharpless et al., 2004; Serrano et al., 1996). The synergism of dual p16INK4a and ARF loss on mouse tumorigenesis is consistent with the distinct molecular functions of each protein and with human clinical data showing their frequent co-inactivation. Still, it was disappointing that mice lacking ARF, p16INK4a, or both factors did not frequently develop the types of cancer commonly seen in humans bearing INK4a/ARF alterations (e.g., melanoma, glioblastoma, and pancreatic adenocarcinoma). Most mice lacking ARF and/or p16INK4a develop sarcomas or

lymphomas. It is widely appreciated that human tumors sustain multiple genetic changes concomitant with INK4a/ARF loss (Hanahan and Weinberg, 2011). Therefore, many studies have since tested the cooperative effects of INK4a and/or ARF loss when coupled with oncogene activation (e.g., Ras or Myc) or tumor suppressor inactivation (e.g., p53) (see Table 1). Such compound mouse mutants successfully model the tumor types seen in humans and demonstrate a critical role for p16INK4a and/or ARF inactivation in those cancers. Conversely, ‘super-INK4a/ARF’ mice expressing one or

452

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus

two extra alleles of the INK4a/ARF/INK4b locus display a dose-dependent resistance to cancer (Matheu et al., 2007, 2009).

ARF, a Multi-Faceted Tumor Suppressor Unlike p16INK4a, which has one target (Cdk4/6) and acts within a single pathway, ARF binds to many proteins and regulates numerous cancer pathways (Figure 4), as reviewed in (Sherr, 2006; Ozenne et al., 2010; Maggi et al., 2014; Gil and Peters, 2006). The dominant mechanism by which ARF prevents cancer is through activation of p53. Widely considered the ‘guardian of the genome,’ p53 is the most commonly inactivated tumor suppressor gene in human cancers (Levine and Oren, 2009; Vousden and Prives, 2009). As a transcription factor, p53 regulates over a hundred genes involved in cell proliferation, survival, senescence, metabolism, and migration, among other processes in response to cellular stress. In so doing, p53 activates checkpoints that protect cells from hypoxia, telomere attrition, oncogene activation, DNA damage, and other insults that threaten the genome. It is normally kept at low levels in cells by Mdm2, an E3 ubiquitin ligase and p53 transcriptional target that promotes p53 ubiquitination and degradation (Senturk and Manfredi, 2012). Mdm2 can also block p53 transactivation and facilitate its translocation out of the nucleus. ARF inhibits Mdm2 and disrupts the Mdm2–p53 negative feedback loop, thereby stabilizing and activating p53. It does so by binding Mdm2 in either the nucleoplasm or nucleoli, physically sequestering Mdm2 away from p53 while also antagonizing Mdm2 ubiquitin ligase activity. As a result, ARF potently stimulates p53-dependent transcription and growth inhibition.

Oncogene activation, DNA damage, aging signals

ARF

p53 independent pathways ARF Mdm2 Inactive

+

p53

NPM Myc Mdm2 MdmX/4 HPV E7 TBP1

E2F1/DP1 ATM/ATR CTBP1/2 HIF1 FoxM1b NIAM

E2F4 BCL6 DDX5 UBC9 Topol etc ...

Senescence, cell cycle arrest, apoptosis, DNA repair, etc... Figure 4 The ARF anti-proliferative pathways. ARF expression is induced by aberrant hyper-proliferative signals elicited by activated oncogenes or events during cellular aging, including DNA damage, that lead to INK4a/ARF derepression. Once expressed, ARF activates p53-mediated transcription through its inhibition of Mdm2. ARF also binds numerous proteins independently of p53 and consequently regulates many other cancer pathways. Through both p53-dependent and p53-independent mechanisms, ARF promotes senescence, cell cycle arrest, apoptosis, and DNA repair, among other antitumor activities, thereby helping to preserve genomic integrity.

Early models suggested that ARF acted solely through p53 to suppress cancer. This was based on initial studies suggesting mutually exclusive loss of ARF or p53 in human tumors, as well as observations that ARF caused robust cell cycle arrest and death in p53-positive (but not p53-negative) cells. However, many studies over the years have shown that ARF retains substantial tumor suppressive activity in the absence of p53 (Sherr, 2006; Ozenne et al., 2010; Maggi et al., 2014). Specifically, ARF expression can cause delayed apoptosis, cell cycle arrest or senescence in cells with nonfunctional p53. It can also inhibit ribosome biogenesis, reduce tumor cell migration and angiogenesis, activate pathways that repair damaged DNA, and promote autophagy (‘self-eating’). For the latter, full-length ARF and a short mitochondrial ARF (smARF; translated from an internal methionine) isoform have been shown to localize to mitochondria and induce autophagy in a p53- and caspaseindependent manner (Balaburski et al., 2010). Collectively, these p53-independent activities of ARF reflect its binding to numerous proteins besides Mdm2. Approximately 40 ARF partners have been identified to date (Figure 4). There is still much to learn about the biological significance of ARF’s many interactions. Nevertheless, the diversity of ARF binding proteins underscores the breadth of ARF’s p53dependent and p53-independent tumor suppressive activities (Sherr, 2006; Maggi et al., 2014; Ozenne et al., 2010). ARF associates with transcriptional activators (e.g., Myc, HIF1α, FoxM1b, E2F1, DP1, YY1, p53, and p63), transcriptional repressors (e.g., E2F4, BCL6, CtBP1/2), checkpoint kinases (e.g., ATM and ATR), ligases involved in ubiquitination or sumoylation (e.g., Mdm2, ARF-BP1, and UBC9), factors controlling ribosomal RNA and DNA synthesis (e.g., NPM, nucleolin, TTF-1, DDX5, and UBF), DNA modifying enzymes (e.g., WRN and Topoisomerase I (TopoI)), acetyltransferases (e.g., Tip60), and viral proteins (e.g., TBP1 and human papilloma virus (HPV) 16 E7), among other proteins. Many of those partners are oncogenic proteins, such as Myc and FoxM1b, whose tumor-promoting activities are inhibited by ARF. Other partners suppress cancer (e.g., Tip60 and ATM) and are activated by ARF as part of checkpoints that protect against hyper-proliferative signaling (i.e., oncogene activation) or DNA damage. Notably, cooperative effects of ARF’s p53-dependent and p53-independent activities are expected since many ARF partners affect p53 signaling while also influencing other cellular processes separate from p53. Mdm2 and its relative, MdmX (also called Mdm4), for example, both inhibit p53 yet can also function independently of p53 in the DNA damage response to impede DNA repair and increase genome instability (Melo and Eischen, 2012; Senturk and Manfredi, 2012). Similarly, NIAM is a 'nuclear interactor of ARF and Mdm2' that is able to activate p53 through inhibition of Mdm2 (Reed et al., 2014a), yet it also acts through undefined mechanisms to suppress proliferation and promote chromosomal stability in the absence of p53 (Tompkins et al., 2007). Perhaps the best example is nucleophosmin (NPM1), one of the better validated ARF interacting proteins (Grisendi et al., 2006). NPM1 is an abundant nucleolar phosphoprotein that inhibits ARF-mediated p53 activation by retaining ARF in nucleoli and blocking its sequestration of Mdm2 away from p53. Independently of p53, NPM1 is also required for ARF stability and nucleolar

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus localization (where most ARF protein resides) while ARF upregulation blocks NPM1 nucleocytoplasmic shuttling, thereby interfering with ribosome nuclear export (Sherr, 2006; Maggi et al., 2014). ARF–NPM1 co-regulation therefore impacts p53 signaling as well as other pathways affecting ribosome function and cell proliferation. The physiological importance of ARF’s many p53-independent activities has been demonstrated by the striking phenotypes of mouse tumor models specifically lacking ARF on a p53-deficient background (see Table 1). The first realization that ARF suppresses cancer in living animals independent of p53 came from a seminal study showing that ARF/Mdm2/p53 triple knockout mice develop a wider range of tumor types and multiple primary tumors per animal relative to ARF-/-, p53-/- or Mdm2/p53-/- mice (Weber et al., 2000). Others showed that ARF (but not p53) is required for oncogene-induced senescence in benign nevi, and its loss combined with N-Ras activation promotes melanocyte transformation and melanoma development (Ha et al., 2007). In a mouse model of pancreatic neuroendocrine tumors in which p53 and RB1 are both inactivated by the SV40 large T antigen, ARF deficiency accelerates tumor formation by promoting tumor angiogenesis (Ulanet and Hanahan, 2010). These in vivo studies exemplify how ARF can suppress cancer independent of p53 through multiple mechanisms, such as promoting senescence in benign neoplasias and inhibiting blood vessel formation within existing tumors.

Regulation and Loss of INK4a/ARF in Cancer Genetic inactivation of INK4a/ARF is a frequent event in tumor development, observed in nearly half of all human malignancies. However, functional loss of p16INK4a and/or ARF in tumors can be achieved through a variety of other mechanisms

G1 arrest senescence

including enhanced promoter methylation and silencing, loss of transcriptional activators, reduced translation, increased protein degradation, and altered expression of their target/effector proteins (Figure 5). In fact, alterations of INK4a/ARF or its regulators and effector pathways are thought to occur in essentially all human cancers. In many cases, the entire locus is lost by genetic deletion or coordinated transcriptional repression; however, the separate control of INK4a and ARF by unique factors or signals reveals how a significant number of tumors with an intact INK4a/ARF locus may lack ARF function while retaining p16INK4a activity (or vice versa). In normal cells, INK4a/ARF is tightly silenced by chromatin-associated Polycomb repressive complexes (PRC1 and PRC2) and histone deacetylase (HDAC) complexes (Gil and Peters, 2006; Kim and Sharpless, 2006; Aguilo et al., 2011). This is important for normal cell proliferation, stem cell selfrenewal, and tissue homeostasis since aberrant derepression of INK4a/ARF (as seen in mouse knockouts lacking PRC components Bmi1, CBX7 or EZH2) causes significant hematological, pancreatic, and neurological defects. PRC recruitment to INK4a/ARF is mediated by binding of the Polycomb group protein, CBX7, to the long noncoding antisense RNA ANRIL (Yap et al., 2010), which spans the INK4b/INK4a/ ARF locus (see Figure 2). As normal cells senesce or encounter stresses that induce senescence, such as oncogene activation or DNA damage, the expression of ANRIL and EZH2 declines leading to displacement of the repressive complexes and activated transcription of INK4a/ARF. The consequent induction of p16INK4a and ARF signaling provides a powerful defense against cellular transformation and tumor progression. In cancer cells, however, the levels of ANRIL, CBX7, and other PRC components can be elevated causing improper maintenance of INK4a/ARF repression. Induction of INK4a/ARF is principally stimulated by hyperproliferative signals emanating from activated oncogenes, such

Negative regulators

Positive regulators

PcG protiens (Bmi1, CBX7, EZH2), RB1, ANRIL, E2F3b, Twist*, JunD*, AML-ETO, Tbx2/3, ID1/3#, p53*, ULF*, HuR, ARF#

Activated oncogenes (Ras, Myc, Abl, E1A, PDGFR, EGFR, etc), E2F1, E2F3a, JunB#, DMP-1*, nucleolin*, Ets 1/2#

E2F-mediated S phase entry

RB1

D/K4 D/K6

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INK4a OR ARF Functional loss INK4a/ARF Genetic inactivation

Mdm2

p53

Other ARF partners and pathways

Proliferation (G1,S,G2 arrest) Senescence Apoptosis Migration Metabolism Angiogenesis DNA repair Autophagy Ribosome biogenesis

Figure 5 Multiple targets for disruption of p16INK4a and ARF function in human cancers. In normal cells, the INK4a/ARF locus is tightly silenced by repressive complexes containing Polycomb group (PcG) proteins, such as Bmi1, CBX7, and EZH2. Derepression of the locus is induced by hyper-proliferative signals emitted by activated oncogenes, such as Ras or Myc. This leads to p16INK4a and ARF expression, activation of their respective tumor suppressor pathways, and induction of senescence or apoptosis (acute response) or reduced cell proliferation, DNA repair, migration, metabolism, etc. Most cells maintain this ‘inhibited state.’ However, the few cells that escape those constraints can do so through a variety of mechanisms: (1) genetic deletion or mutation of INK4a/ARF, (2) promoter silencing due to upregulation of factors in the PcG repressive complexes and DNA hyper-methylation, (3) loss of key activators (e.g., the DMP-1 transcription factor for ARF) or effectors (e.g., RB1 for p16INK4a or p53 for ARF), and (4) hyper-activation of oncogenic target proteins (examples are bolded). An incomplete listing of negative and positive regulators of INK4a/ARF is shown, with specific regulators of ARF (*) and p16INK4a (#) indicated.

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as Ras or Myc, thereby engaging RB1, p53, and ARF/p53-independent checkpoints that protect against unrestrained cell proliferation, genomic mutation, and cancer (Kim and Sharpless, 2006; Gil and Peters, 2006; Ozenne et al., 2010; Sherr, 2012). For the most part, oncogenic stress causes the coordinated upregulation of both p16INK4a and ARF by transcriptional activators, such as E2F1. The role of INK4a/ARF in DNA damage checkpoints is less clear and more controversial. Various types of DNA damage induce p16INK4a, ARF, or both proteins; however, their upregulation is generally delayed and may be more important for mediating long-term responses (including DNA repair or senescence) following chronic and/or low-level DNA damage as opposed to acute DNA damage. Consistent with that notion, ARF was found to be required for p53-dependent tumor suppressive activities in irradiated mice once the acute, pathological response to DNA damage had ended (Christophorou et al., 2006). While INK4a and ARF are often co-regulated, a large number of factors selectively regulate the individual genes or transcripts. Several INK4a-specific transcriptional activators (e.g., JunB, Ets1/2) and repressors (e.g., Id1, Id3) have been identified, as have ARF-specific transcriptional activators (e.g., DMP-1, AML1) and repressors (e.g., Twist, AML–ETO). At the posttranscriptional level, the RNA binding protein HuR (which is upregulated in various cancers and inhibits senescence) negatively regulates INK4a and ARF mRNAs but through distinct mechanisms that depend on cell context (Maggi and Weber, 2013). In human diploid fibroblasts, HuR binds and destabilizes the INK4a mRNA whereas in MEFs it binds the 5′ UTR of the ARF mRNA and blocks its translation by preventing nucleolin-ARF mRNA association and export to the cytoplasm. The basis for differential HuR regulation of INK4a and ARF in mouse and human cells is unclear, but it is not entirely unusual since Ras activation selectively induces p16INK4a in human cells despite upregulating both p16INK4a and ARF in rodent cells (Gil and Peters, 2006; Kim and Sharpless, 2006). Other mechanisms distinctly controlling ARF or p16INK4a may play an important role in cancer. ULF, an ‘ubiquitin ligase for ARF,’ is upregulated in tumors and lowers ARF levels by promoting its lysine-independent ubiquitination and proteasomal degradation (Chen et al., 2010). Intriguingly, ARF can promote p16INK4a degradation via its interaction with the REGγ subunit of the proteasome (Kobayashi et al., 2013). Such crosstalk between the INK4a/ARF products may constitute an autoregulatory feedback loop to reduce p16INK4a levels, enabling cell cycle reentry following a successful checkpoint response and thereby delaying aging (see below). ARF is also subject to negative feedback regulation but it occurs at the transcriptional level due to p53-mediated repression. Specifically, ARF is induced by activated oncogenes and activates p53; subsequently, p53 binds the promoter for ARF (not INK4a) and recruits HDACs and Polycomb group proteins to repress its transcription (Robertson and Jones, 1998; Zeng et al., 2011). Those results explain why cancer cells lacking p53 often express high levels of ARF. Increased expression of ARF in cells lacking p53 is a beneficial barrier against tumorigenesis due to its many p53independent activities, as discussed earlier. This was effectively illustrated in a recent study that showed ARF upregulation

following acute p53 loss inhibits interferon β (IFN-β) production and signaling, markedly restricting the proliferation and tumorigenicity of those cells (Forys et al., 2014). Unfortunately, there is strong selective pressure for cells to escape such constraints and remove ARF to enable proliferation, facilitating the outgrowth of tumor cells with activated IFN-β signaling. In fact, analyses of human triple-negative breast cancer samples revealed that more than 50% have co-inactivation of ARF and p53 as well as hyperactive IFN signaling. Those findings may be useful in the clinic since they suggest that therapies targeting IFN-β effectors, such as STAT1, would be appropriate for treating tumors with combined p53 and ARF inactivation.

INK4a/ARF in Aging Genome-wide association studies (GWAS) in millions of patients implicate the INK4a/ARF locus in a range of aging-related diseases (Jeck et al., 2012). The data identify a hotspot of single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 spanning the INK4a/ARF locus (and the sequences encoding p15INK4b and ANRIL) that is associated with increased risk for type 2 diabetes, glaucoma, late onset Alzheimer’s, frailty, and atherosclerotic pathologies including stroke, myocardial infarction, and aortic aneurysm (see Figure 2). Exactly how the different SNPs affect ANRIL and INK4a/ARF expression, however, is not well understood. Some SNPs associated with a higher risk of atherosclerosis increase the expression of ANRIL isoforms and circular species, thereby enhancing repression of INK4a/ARF in peripheral blood T cells and causing reduced p15INK4b, p16INK4a, and ARF levels (Burd et al., 2010). Other SNPs may reduce ANRIL expression, which would be predicted to increase p16INK4a and ARF levels. That seems likely for SNPs associated with type 2 diabetes since precocious expression of p16INK4a and ARF in pancreatic β cells of EZH2-null mice impairs β cell proliferation, reduces insulin production, and promotes type 2 diabetes (Chen et al., 2009). Rescue of those defects by INK4a/ ARF deletion established a causative role for p16INK4a and/or ARF in that process. The observations above suggest that loss and gain of INK4a/ARF expression can each promote aging phenotypes, so does the locus accelerate or delay aging? The answer is complicated and still unfolding. On the one hand, substantial evidence suggests INK4a/ARF enhances aging (reviewed in Sorrentino et al., 2014; Sherr, 2012). This includes findings that p16INK4a and ARF levels increase in aging mouse and human tissues, promote cellular senescence (which accumulates with organismal aging), and are regulated by age-modifying stimuli such as caloric restriction. Also, elevated p16INK4a and ARF levels have been linked to the agedependent decline in normal stem cell function in the neural, pancreatic, muscle, and hematopoietic stem cell compartments. On the other hand, transgenic mice expressing two extra copies of the INK4b/ARF/INK4a locus (INK4/ARF-tg/tg) have reduced signs of aging and a longer life span, with the delayed aging phenotype distinct from the reduced cancer incidence seen in those animals (Matheu et al., 2009). How can such apparently contradictory findings be reconciled?

Cell Division/Death: Cell Cycle: The INK4a/ARF Locus One explanation is that p16INK4a and ARF have separate, opposing roles in aging and that the pro-aging effects of p16INK4a may be countered by antiaging activities of ARF. For instance, p16INK4a (not ARF) expression is increased in aging T-cell progenitors, significantly reduces their number and promotes thymic involution (Berent-Maoz et al., 2012), a wellrecognized change in the elderly linked with impaired immunity. Also, selective ablation of INK4a or ARF in a progeroid mouse model of premature aging (BubR1-deficient mice) showed that p16INK4a promotes senescence and aging, whereas ARF suppresses those processes (Baker et al., 2008, 2011). The antiaging effect of ARF in this setting may be partly mediated through negative regulation of p16INK4a levels since ARF deletion increases p16INK4a expression in tissues displaying accelerated aging (muscle, fat, and eye). ARF may also protect against aging by inducing a p53 transcriptional response involving antioxidant gene expression which promotes quiescence, DNA repair, and cell survival. The fact that p53 loss phenocopies ARF loss in BubR1 mutant mice supports that notion (Baker et al., 2008), as does the upregulation of antioxidant p53 targets and delayed aging of ARF/p53 transgenic mice (Matheu et al., 2007). It is also possible that both INK4a and ARF have antiaging effects although the timing and context of their expression may be important. Although acute INK4a/ARF upregulation during oncogenesis may cause excessive cell apoptosis or senescence that compromises tissue integrity and accelerates aging, the progressive low-level activation of the locus during aging could be beneficial by slowing cell proliferation rates or triggering a reversible quiescent arrest, thereby delaying the exhaustion of stem cell pools (Matheu et al., 2009). The mitochondrial smARF form of ARF may also inhibit aging. Studies in neurons showed that smARF is a critical upstream activator of a Parkin/PINK1 mitophagy pathway that eliminates damaged mitochondria from cells (Grenier et al., 2014). This is relevant because impaired Parkin/PINK1-mediated mitophagy in neurons is thought to cause the accumulation of damaged mitochondria that occurs during aging and is associated with Parkinson’s disease (PD). Whether or not smARF levels and activity are impaired in PD brains and how this pathway contributes to neurodegeneration in PD remains to be determined. Indeed, more work is needed to define the individual and combined roles of p16INK4a, ARF, and smARF in aging and age-related disorders. Nonetheless, the cumulative evidence establishes that INK4a/ARF is a bona fide aging locus.

Unique Roles of ARF in Development INK4a/ARF repression is necessary and widespread throughout most tissues during development. However, there are a few cell types where transient ARF expression is critical for proper development, particularly in the eye and testes (Gromley et al., 2009). ARF-null mice are blind with smaller eyes than ARFpositive animals, mimicking a human eye disease called persistent hyperplastic primary vitreous (PHPV) (McKeller et al., 2002). The blindness results from elevated expression of platelet-derived growth factor receptor β (PDGFRβ) in perivascular cells within the vitreous of the eye, which causes their

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aberrant proliferation and prevents the regression of the hyaloid vascular system required for proper retina and lens formation (Reed et al., 2014b). At the molecular level, ARF suppresses PDGFRβ expression through complementary mechanisms that are p53-dependent (repression of PDGFRβ transcription) and p53-independent (upregulation of the miR34a microRNA and inhibition of PDGFRβ translation). Paradoxically, ARF expression in male germ cells has no effect on proliferation and instead promotes the survival of spermatogonia by blocking p53-mediated apoptosis, contrasting with its role as a p53 activator in cancer (Churchman et al., 2011). INK4a-null mice do not display developmental defects. However, it is notable that overexpression of p16INK4a, p15INK4b, and ARF in transgenic INK4/ARF-tg/tg mice causes male sterility (see Table 1; Matheu et al., 2009). This is thought to reflect a proliferative defect of the spermatogonia due to increased INK4a/b-RB1 mediated growth inhibition rather than ARF-p53 effects.

Conclusions In summary, ARF and p16INK4a are major barriers to the outgrowth of cancer cells. Their induction by various cell stresses, particularly hyper-proliferative stimuli and aging signals, and consequent activation of the p53 and RB1 tumor suppressors is critical for their anticancer function. In the absence of p53, ARF can also function through many other pathways to induce senescence and suppress tumor cell survival, migration, angiogenesis, etc. Through those pathways, each protein engages protective checkpoints that inhibit cancer cell outgrowth by reversible cell cycle arrest (and DNA repair) of mildly damaged cells or irreversible removal of severely damaged cells via senescence or apoptosis, thereby ensuring genomic integrity. The importance of these pathways in human cancer is demonstrated by their frequent disruption in tumors. Indeed, functional inactivation of p16INK4a, ARF, p53, and/or RB1 is thought to occur in most, if not all, human tumors due to genetic/epigenetic inactivation and alterations targeting their signaling pathways (Figure 5). Advancing our understanding of these pathways and employing that knowledge to improve tumor diagnosis, prognosis, and treatment is the ultimate goal. For example, preclinical studies suggest that ARF peptides targeting the FoxM1b oncogenic protein have beneficial antitumor activity in mouse models of lymphoma and sarcoma (Wang et al., 2013). Selective inhibitors of Cdk4/6 are further along in development and show significant clinical promise in treating RB1-positive tumors that lack functional p16INK4a (Flaherty et al., 2012). Derepression of the INK4a/ARF locus is also important in cellular and organismal aging although its impact on age-related pathologies and longevity is complex. Whether or not INK4a/ARF is a benefit or liability to aging is controversial and still under investigation. The context (timing, level, cause, and tissue) of INK4a/ARF regulation and whether p16INK4a, ARF or both proteins are expressed may be critical in dictating outcome. That said, the fact that increased INK4/ARF gene dosage in mice leads to delayed aging and an extended life span (as well as increased cancer resistance) makes a

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persuasive case that more INK4a/ARF is better, at least if you are a mouse (and female, since the male transgenic mice are sterile). For humans, any potential benefit of manipulating INK4a/ARF to prolong life (beyond the protection against cancer) requires a firmer grasp on how, when, and where p16INK4a and/or ARF expression influences the aging process.

See also: Nucleic Acid Synthesis/Breakdown: RNA Synthesis/ Function: Comparison of Bacterial and Eukaryotic Replisome Components

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Relevant Websites http://www.cancer.gov/clinicaltrials/search/resultsprotocolsearchid=6184393 List of NCI-sponsored clinical trials involving the use of selective Cdk4 and Cdk6 inhibitors, such as palbociclib (PD-0332991), to treat various malignancies. http://www.cbioportal.org/ The cBioPortal for Cancer Genomics. http://cancergenome.nih.gov Website for the Cancer Genome Atlas (TCGA).